Classical Complement Pathway ELISA Kit [Human] (CAT#: CB-P059-K) Datasheet

Product Type
Kit
Description
The complement system plays an essential role in chronic, autoimmune and infectious disease. There are three pathways of complement activation, namely the classical, the alternative and the lectin pathway. Impaired complement activity causes humans to become susceptible to repetitive fulminant or severe infections and may contribute to development of autoimmune disease. Inappropriate activation of complement contributes to chronic inflammation and tissue injury. In vitro activation of the complement sequence leads to the consumption of complement components which, in turn, leads to a decrease in their concentration. Thus, the determination of complement proteins or complement activity is used to indicate whether the complement system has been activated by an immunologic and/or pathogenic mechanism. Both functional and immunochemical complement measurements are used to evaluate subjects when a complement-activating disease is suspected or an inherited deficiency is possible. The level of complement activity evaluated by functional assays such as Complement kit takes into account the rate of synthesis, degradation, and consumption of the components and provides a measure of the integrity of the pathways as opposed to immunochemical methods which specifically measure the concentration of various complement components.
Applications
This kit is an enzyme immunoassay (ELISA) used to determine the functional classic complement pathway in human serum. For research use only, not for diagnostic procedures.
Principle
The Complement Classical pathway Kit combines principles of the hemolytic assay for complement activation with the use of labelled antibodies specific for neoantigen produced as a result of complement activation. The amount of neoantigen generated is proportional to the functional activity of complement pathways. In the Complement CP kit, the wells of the microtitre strips are coated with specific activators of the classical pathway. This in combination with sample dilution buffer composition and serum dilution level ensure that only the Classical pathway is activated. During the incubation of the diluted serum in the wells, complement is activated by the specific coating. The wells are then washed and the amount of C5b-9 complex formed on the plate surface is detected Alternative pathway (C3, factor D, B, P) C3 C3b C3a Lectin pathway (MBL, MASP -2, C4, C2) Classical pathway (C1q, C1r, C1s, C4, C2) C4b2a C4b2a C3bBbP Terminal pathway (C5, C6, C7, C8, C9 C5b-C9 (MAC) COMPL CP310 RUO, LABEL-DOC-0030, 3.0 3 with a specific alkaline phosphatase labelled antibody to the C5b-9 neoantigen formed during MAC (Membrane Attack Complex) formation. After a further washing step, detection of specific antibodies is obtained by incubation with alkaline phosphatase substrate solution. The amount of complement activation correlates with the colour intensity and is measured in terms of absorbance (optical density (OD).
Detection Method
Enzyme immunoassay (ELISA) technique
Sample
Human serum.
Sample Volume
100 μL of properly diluted (1:101) unknown / determination.
Controls
1 negative control, ready to use.
1 positive control, lyophilized.
1 activity control, lyophilized.
Species Reactivity
Human
Protocol Length
1 hour incubation (37°C) + 30 min. (RT) + 30 min. (RT) = 2 hours total incubation time.
Components
-One frame with blue coloured break-apart wells (12x8) coated with human IgM
- 2 x 35 mL Diluent CP (Dil CP), labelled blue.
- 13 mL conjugate containing alkaline phosphatase-labelled antibodies to C5b-9 (blue colour).
- 13 mL Substrate solution ready to use.
- 30 mL wash solution 30x concentrated.
- 0.2 mL negative control (NC) containing human serum (to be diluted as for a subject serum
sample).
- Lyophilized positive control (PC) containing freezed dried human serum, to be reconstituted in
0.2 mL distilled water, see "Reconstitution of positive control", below.
- Lyophilized activity control (AC) for semi-quantitative application, containing freezed dried human serum (different origin than PC).U14
Requires
-Microplate reader with filter 405 nm.
- Precision pipettes with disposable tips.
- Washer for strips, absorbent tissue, tubes and a timer.
Handling Advice
All reagents in the kit are ready for use except washing solution and controls. The reagents should be
stored at 2-8°C except the positive control. The positive control must be stored at -21°C.
Storage
The reagents should be stored at 2-8° C except the positive control and activity control. The positive control and activity control should be stored at -20°C.
Research Use
For research use only. Not for use in or on humans or animals or for diagnostics. It is the responsibility of the user to comply with all local/state and Federal rules in the use of this product. Hycult Biotech is not responsible for any patent infringements that might result with the use of or derivation of this product.

Figure 1.

Table 1.

Table 2.
Sera with known complement deficiencies were tested in the assay and the following results were obtained. All deficient sera were detected in the assay and gave values below 5 %**).

Table 3. Inter-assay precision for qualitative application was determined by testing three samples in duplicate. Results were obtained for six different runs.

Table 4. Intra-assay precision for qualitative application was determined by testing one sample in 40 wells.

All products and services are for Research Use Only. Do Not use in humans.

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